The focus of the research in my laboratory is the eukaryotic nucleus: its structure and function. We work both in vivo and in vitro. For in vivo studies, we use mammalian cultured cell lines to examine nuclear transport or assembly using RNAi technology to knock down expression of genes of interest to determine their effect on function. For in vitro studies, we use a nuclear reconstitution system which efficiently assembles nuclei in the test tube from soluble and membrane components. This system makes use of an extract of Xenopus eggs, which naturally store abundant amounts of disassembled nuclear components in preparation for the rapid cleavages of early division. Upon addition of DNA or chromatin to the in vitro extract, nuclei containing nuclear membranes, a nuclear lamina, and nuclear pores quickly assemble. The in vitro reconstituted nuclei are capable of nuclear import, DNA replication, pol III transcription, and even mitotic disassembly.
A major interest of the laboratory is the nuclear pore, a large macromolecular complex of 120 million daltons which spans the nuclear membranes. All communication between the nucleus and cytoplasm occurs through this nuclear pore complex. In vivo, the pore actively imports nuclear proteins, while exporting mRNA, tRNA, snRNA, and ribosomal precursors. We study: (1) assembly of the nucleus itself, (2) assembly of nuclear pores, (3) the regulation of these processes, and (4) the mechanism of nuclear transport, including mRNA export, protein export, and protein import. Using antibodies we can immunodeplete the assembly extract of individual nuclear pore proteins, then reconstitute nuclei which lack that protein. Such "designer" nuclei can be tested for alterations in nuclear pore structure, pore function, or pore assembly. We recently discovered the protein responsible for initiating pore assembly at the nucleus (Rasala et al, 2006, 2008). This protein, ELYS, acts by binding to chromatin at AT-rich sequences and recruiting the key structural subunit of the nuclear pore to these sites to initiate nuclear pore assembly. With experiments of this type, we hope to molecularly dissect and elucidate the gates that "guard the fortress" of the genome.
A second major interest of the laboratory involves understanding assembly of the eukaryotic nucleus itself. Specifically, we are focused on how nuclear assembly is regulated such that nuclear membranes form around chromatin in a spatially precise manner and, once formed, how the nuclear membranes acquire nuclear pores. We have shown that an abundant cellular protein, importin beta, acts in mitosis to negatively regulate both nuclear membrane fusion and nuclear pore assembly (Harel et al, M.B.C, 2003). The small GTPase Ran acts as the counteracting positive regulator. We believe that importin beta acts as a global regulator of cellular events involving the genome, including those controlling nuclear import, spindle formation, correct nuclear membrane assembly, and nuclear pore assembly.
Harel, A., Orjalo, A., Vincent, T., Lachish-Zalait, A., Vasu, S., Shah, S., Zimmerman, A., Elbaum, M. and Forbes, D.J. (2003). Removal of a Single Pore Subcomplex Results in Vertebrate Nuclei Devoid of Nuclear Pores. Molecular Cell 11:853-864.
Harel, A. , Chan, R. C., Lachish-Zalait, A., Zimmerman, A., Elbaum, M., and Forbes, D. (2003). Importin beta negatively regulates nuclear membrane fusion and NPC assembly. Molecular Biology of the Cell 14:4387-4396.
Harel, A. and D. J. Forbes. (2004). Importin beta: Conducting a much larger cellular symphony. Molecular Cell 16:319-330.
Orjalo, A.V., Arnaoutov, A. , Shen, Z., Boyarchuk, Y., Zeitlin, S.G., Fontoura, B., Briggs, S., Dasso, M., and D.J. Forbes. (2006). The Nup107-160 Nucleoporin Complex Is Required for Correct Bipolar Spindle Assembly. Molecular Biology of the Cell 17:3806-3818.
Rasala, B.A., Orjalo, A.V., Shen, Z., Briggs, S., and D.J. Forbes. (2006). ELYS is a Dual Nucleoporin/Kinetochore Protein Required for Nuclear Pore Assembly and Proper Cell Division. Proc. Natl. Acad. Sci. U.S.A. 103:17801-17806.
Resendes, K. , Rasala, B., and D. J. Forbes. (2007). Centrin 2 Localizes to the Vertebrate Nuclear Pore and Plays a role in mRNA and Protein Export. Molecular and Cellular Biolog, 28:1755-1769.
Brown, C., Kennedy, C., Delmar, V., Forbes, D.J. and P. Silver. (2008). Global Histone Acetylation Induces Functional Genomic Reorganization at Mammalian Nuclear Pores. Genes and Development 22 627-639.
Chakraborty, P., Wang, Y., Wei, J-H. , van Deursen, J., Yu, H., Malureanu, L., Dasso, M., Forbes, D.J., Levy, D.E., Seemann, J. , and B. M. A. Fontoura. (2008). Nucleoporin Levels Regulate Cell Cycle Progression and Phase-Specific Gene Expression. Developmental Cell 15:657-667.
Rasala, B. A., Ramos, C., Harel, A., and , D. J. Forbes. (2008). Capture of AT-rich Chromatin by ELYS Recruits POM121 and NDC1 to Initiate Nuclear Pore Assembly. Molecular Biology of the Cell 19:3982-3996.
Douglass Forbes received her Ph.D. from the University of Oregon and was a postdoctoral fellow at UCSF in the Dept. of Biochemistry and Biophysics. She was a PEW Fellow in the Biomedical Sciences and on the Governing Council of the American Society of Cell Biology. She is presently Vice Chair of the Section of Cell and Developmental Biology at UCSD.